Diversity and Evolution of Francisella tularensis Gunnell et al. The Genetic Diversity and Evolution of Francisella tularensis with Comments on Detection by PCR Mark K. Gunnell1,3*, Byron J. Adams2 and Richard A. (McCoy and Chapin, 1912). Following the Bacterium Robison1 genus, it was subsequently placed in Pasteurella and later Brucella (Salomonsson, 2008). Finally in 1959, it was 1Department of Microbiology and Molecular Biology, placed in a new genus, Francisella, in honor of Edward Brigham Young University, Provo, UT 84602, USA Francis, in which genus it resides today (Olsufjev et al., 2Department of Biology, and Evolutionary Ecology Laboratories, 1959). There are currently four recognized subspecies of Brigham Young University, Provo, UT 84602, USA Francisella tularensis: tularensis, holarctica, mediasiatica, 3Microbiology Branch, Life Sciences Division, Dugway and novicida. While the inclusion of novicida as a Proving Ground, Dugway, UT 84022, USA subspecies of F. tularensis is still contested (Larsson et al., *Corresponding Author: [email protected] 2009; Kingry and Petersen, 2014), much of the recent scientific literature, including Bergey’s Manual of http://dx.doi.org/10.21775/cimb.018.079 Systematic Bacteriology, recognizes this classification (Garrity, 2005). Abstract Francisella tularensis has been the focus of much research In 1950, the first novicida subspecies was isolated and over the last two decades mainly because of its potential characterized (Larson et al., 1955). This new isolate use as an agent of bioterrorism. F. tularensis is the resembled F. tularensis morphologically, but differed in that causative agent of zoonotic tularemia and has a worldwide it could ferment glucose, was not as virulent in humans, distribution. The different subspecies of F. tularensis vary in and did not cross-react with serum from rabbits inoculated their biogeography and virulence, making early detection with killed F. tularensis. Based on these differences, the and diagnosis important in both the biodefense and public authors proposed the name Francisella novicida (Larson et health sectors. Recent genome sequencing efforts reveal al., 1955). However, in the 1950s, researchers did not have aspects of genetic diversity, evolution and phylogeography the genetic tools which became available in later decades. previously unknown for this relatively small organism, and In the 1980s, DNA-DNA hybridization experiments between highlight a role for detection by various PCR assays. This F. tularensis and F. novicida demonstrated up to 92% review explores the advances made in understanding the homology (Hollis et al., 1989). Because of this high degree evolution and genetic diversity of F. tularensis and how of genetic similarity, it was proposed that F. novicida be these advances have led to better PCR assays for reclassified as a subspecies of F. tularensis . This detection and identification of the subspecies. reclassification was formally proposed in 2010 in the International Journal of Systematic and Evolutionary Introduction Microbiology (IJSEM) (Huber et al., 2010). This proposal Francisella tularensis is a small, non-motile, Gram-negative received a formal objection in IJSEM, contending that coccobacillus and is the causative agent of the zoonotic genetic similarity was not enough to reclassify F. novicida disease tularemia. This facultative intracellular pathogen as F. tularensis subsp. novicida, but that the phenotypic was first discovered in Tulare County California in 1911 differences were sufficient enough to justify separate where it caused a plague-like illness in local rodents species designation (Johansson et al., 2010). (McCoy and Chapin, 1912). F. tularensis is able to cause disease in rabbits, squirrels, and other mammals, including Finally, in a rebuttal to the objection of Johansson et al., humans (Wherry and Lamb, 1914). The transmission of F. Busse et al. (2010), stood by their initial recommendation for tularensis to humans is mediated through arthropod reclassification, asserting that the genetic similarity meets vectors such as ticks and deer flies, by the ingestion of the definition of a subspecies (Wayne et al., 1987). contaminated food or water, or by inhalation of aerosolized Furthermore, Busse et al. acknowledge the phenotypic bacteria (Akimana and Abu Kwaik, 2011). F. tularensis differences between F. tularensis and F. novicida , but subsp. tularensis is highly infectious. It is estimated that an contend that the 11 phenotypic differences noted are not aerosol inoculation of as few as 10 organisms is sufficient sufficient enough for a new species (Busse et al., 2010). to cause disease in humans (McCrumb, 1961). Because of There are many other examples of bacteria with a greater its highly infectious nature, F. tularensis is considered a percentage of phenotypic differences which are classified as potential agent of bioterrorism and is categorized by the the same species (e.g. the various biovars of Pseudomonas Centers for Disease Control and Prevention (CDC) as a fluorescens) (Busse et al., 2010). Despite this evidence, a Tier 1 select agent (Dennis et al., 2001). formal reclassification has yet to occur. Based on the high genetic similarity, and taking into account the relatively few Through the years, the taxonomy of Francisella has gone phenotypic differences, we also propose the reclassification through many changes. Upon its discovery, McCoy and of F. novicida as a subspecies of F. tularensis , and will refer Chapin named their new discovery Bacterium tularense to it as such throughout this work. Curr. Issues Mol. Biol. (2016) 18: 79-92. horizonpress.com/cimb !79 Diversity and Evolution of Francisella tularensis Gunnell et al. Each subspecies is predominantly associated with a include Type A.I and Type A.II, with the former generally specific geographic distribution and severity of disease. isolated from the eastern United States and the latter The subspecies tularensis is typically found in North generally isolated from the western United States America (Staples et al., 2006) while the subspecies (Johansson et al., 2004). This biogeographic separation is holarctica is found across much of the Northern correlated with the geographic distribution of specific Hemisphere (Johansson et al., 2004). The subspecies vectors, hosts, and other abiotic factors such as elevation mediasiatica has only been isolated from the central Asian and rainfall (Farlow et al., 2005; Oyston, 2008). The major republics of the former Soviet Union (Broekhuijsen et al., divisions of Type B tularensis also display geographic 2003) and the subspecies novicida has been isolated from structure, with Type B.I isolated from Eurasia, Type B.II North America and Australia (Hollis et al., 1989; Whipp et isolated from North America and Scandinavia, Type B.III al., 2003). Phylogenetic relationships among these isolated from Eurasia and North America, Type B.IV subspecies are inferred in Figure 1. isolated from North America and Sweden, and Type B.V isolated from Japan (Johansson et al., 2004). Unlike type A The two subspecies most associated with human disease tularensis, the distribution of Type B tularensis has not are tularensis and holarctica. These are often abbreviated been shown to correlate with the distribution of any specific simply as Type A and Type B tularensis, respectively. Type vectors (Farlow et al., 2005). A tularensis causes a more severe form of tularemia while the presentation of type B tularemia is somewhat milder The F. tularensis subsp. holarctica isolated from Japan was (Owen et al., 1964; Weiss et al., 2007). The subspecies first differentiated from other F. tularensis subspecies mediasiatica is fully virulent in mice, yet is believed to be of based on its ability to ferment glucose (Olsufjev and relatively mild virulence in humans (Broekhuijsen et al., Meshcheryakova, 1983). These isolates were further 2003; Champion et al., 2009). Similar to the subspecies differentiated by demonstrating a reduced virulence from mediasiatica, the subspecies novicida is fully virulent in the subspecies tularensis, displaying a virulence similar to mice, yet rarely causes disease in humans (Hollis et al., that of the subspecies holarctica (Sandstrom et al., 1992). 1989). As genomic tools became more widely available, this division was confirmed by microarray analysis Genetic analyses by multiple-locus variable-number (Broekhuijsen et al., 2003), restriction fragment length tandem repeat analysis (MLVA) has identified further sub polymorphism (RFLP) analysis (Thomas et al., 2003), and classifications and geographic structure of Type A and Type multiple-locus variable number tandem repeat analysis B tularensis. The major subdivisions of Type A tularensis (MLVA) (Johansson et al., 2004; Fujita et al., 2008). Figure 1. Maximum likelihood tree inferring the phylogenetic relationships of the F. tularensis subspecies. Tree was constructed by concatenating 10 housekeeping genes (recA, gyrB, groEL, dnaK, rpoA1, rpoB, rpoD, rpoH, fopA, and sdhA) followed by alignment with Clustal W and generation of the tree with MEGA 5.2. Bootstrap values are indicated at the nodes except where support was less than 0.65. Figure 1. Maximum likelihood tree inferring the phylogenetic relationships of the F. tularensis subspecies. Tree was constructed by concatenating 10 housekeeping genes (recA, gyrB, groEL, dnaK, rpoA1, rpoB, rpoD, rpoH, fopA, and sdhA) followed by alignment with Clustal W and generation of Curr. Issues Mol. Biol. (2016)